Differential expression of “Candidatus Liberibacter solanacearum” genes and prophage loci in different life stages of potato psyllid

Psyllid species, including the potato psyllid (PoP) Bactericera cockerelli (Sulc) (Triozidae) serve as host and vector of “Candidatus Liberibacter spp.” (“Ca. Liberibacter”), which also infects diverse plant hosts, including citrus and tomato. Psyllid transmission of “Ca. Liberibacter” is circulative and propagative. The time of “Ca. Liberibacter” acquisition and therefore vector life stage most competent for bacterial transmission varies by pathosystems. Here, the potato psyllid-“Ca. Liberibacter solanacearum” (CLso) pathosystem was investigated to dissect CLso-prophage interactions in the tomato plant and PoP-psyllid host by real-time quantitative reverse transcriptase amplification of CLso genes/loci with predicted involvement in host infection and psyllid-CLso transmission. Genes/loci analyzed were associated with (1) CLso-adhesion, -invasion, -pathogenicity, and -motility, (2) prophage-adhesion and pathogenicity, and (3) CLso-lysogenic cycle. Relative gene expression was quantified by qRT-PCR amplification from total RNA isolated from CLso-infected 1st–2nd and 4th–5th nymphs and teneral adults and CLso-infected tomato plants in which CLso infection is thought to occur without SC1-SC2 replication. Gene/loci expression was host-dependent and varied with the psyllid developmental stage. Loci previously associated with repressor-anti-repressor regulation in the “Ca Liberibacter asiaticus”-prophage pathosystem, which maintains the lysogenic cycle in Asian citrus psyllid Diaphorina citri, were expressed in CLso-infected psyllids but not in CLso-infected tomato plants.

Psyllids are hemimetabolous insects and have morphologically distinct adult and nymphal (juvenile) or 'immature' stages in which the mature adult stage is preceded by five nymphal stages.Psyllid-mediated transmission of "Ca.Liberibacter spp." utilizes a circulative, propagative mode and once the bacterium is acquired, e.g., associates with the salivary glands and/or oral cavity from which the bacterium is inoculated to plants, or 'transmitted' for the life of the vector 4,5 .However, transmission efficiency varies among the specific psyllid vector and developmental stages, depending on the duration of ingestion and/or salivary gland acquisition 6 and differences in the mechanisms that mediate the pre-acquisition phase of the transmission pathway among the psyllid vectors of CLas and CLso 5 .
As an insect-vectored phytopathogen, "Ca.Liberibacter" spp.have evolved a lifestyle in which they utilize both an animal (insect) and plant host for multiplication 1 .Although speculative, "Ca.Liberibacter" may have

Selection of genes and loci for real-time quantitative RT-PCR analysis
The hypothesis-driven selection of the genes of interest was based on published research results from laboratory and in silico studies 4,8,9 .Thirty-two CLso/CLas bacterial (13) or prophage (20) genes were selected with predicted functions in "Ca.Liberibacter" and prophage pathogenicity, including the potential modulation of PoP host defenses, implicated from previous studies of ACP-CLas and PoP-CLso pathosystems (CLas strain psy62, NC_012985.3)and prophage loci (CLas strain UF506, HQ377374.1;CLso-NZ genome reference (NC_014774.1)(Table 1).Initially, candidate CLas and prophage candidates of interest were identified for the better-annotated CLas-ACP pathosystem with results guiding searches for CLso and prophage candidates.To locate and annotate the CLso orthologs, a homology search was carried out using a protein query for selected gene/loci found in CLas strains psy62 (NC_012985.3) and UF506 (HQ377374.1),and subsequently, the CLso-NZ genome reference (NC_014774.1;CLso-A haplotype) was searched using tblastn and blastx with cut off E-value of ≤ 0.00 and e − 5, respectively.The top 10 BLAST hits for each sequence were considered, and the functional annotations were determined using online bioinformatic tools and/or sequence databases available at the NCBI GenBank website (https:// blast.ncbi.nlm.nih.gov/), UniProtKB-Swiss-Prot (https:// www.expasy.org/ resou rces/ unipr otkb-swissprot), the NCBI Conserved Domain search (https:// www.ncbi.nlm.nih.gov/ Struc ture/ cdd/ wrpsb.cgi), conserved protein domain identification by InterProScan (http:// www.ebi.ac.uk/ Inter ProSc an), and predicted motifs identified by PROSITE Scan (http:// npsa-pbil.ibcp.fr/ cgi-bin/ npsa_ autom at.pl? page= npsa_ prosi te.html).The coding sequence having the highest BLAST similarity score and coverage, together with the most biologically relevant annotation, was further verified by an amino acid homology search for each bacterial chromosomal gene or prophage locus, respectively.
The predicted CLso orthologs for the CLas-associated prophages SC1 and SC2 (HQ377374.1;Zhang et al., 2011) were identified using viroBlast 29 and PHAST (PHAge Search Tool) 30 algorithms with default parameters for homology and prophage region searches, respectively.The orthologs were selected based on the best match among the ten top BLAST hits (viroBlast) having the highest similarity score and coverage with the predicted prophage region of the CLso-NZ genome reference (NC_014774.1),hereafter, "predicted prophage genes" to distinguish them from bacterial CLso chromosomal gene orthologs.
Primers were designed and used to amplify and clone the respective coding regions from total DNA isolated from CLso-A haplotype CLso-infected psyllids, guided by the CDS annotations for CLso-NZ in NCBI (NC_014774.1)genome sequence (Table 1).
Primers for PCR amplification of CLso-prophage genes from infected psyllids were designed using the IDT Primer Quest Primer Design Tool using the default settings.The primer sequences and annealing temperatures are shown in S1  ), 30-90 s extension at 72 °C, and a final extension of 10 min at 72 °C.Amplicons of the expected size were separated by agarose gel (1%) electrophoresis in 1 × Tris-Acetate-EDTA (TAE) buffer, pH 8.0, and bands were visualized by staining with GelRed (Biotium, USA).
Table 1.Predicted bacterial chromosomal genes and CLso-associated prophage loci analyzed in this study.a Loci analyzed for "Candidatus Liberibacter solanacearum" (CLso) haplotype A were designated as "AZph" and "AZch" for predicted bacterial chromosomal genes and prophage loci, respectively.b Selected chromosomal genes corresponding to the "Ca.Liberibacter asiaticus" (CLas) reference, UF506 (HQ377374.1)and psy62 (NC_012985.3),respectively.c Predicted homolog of CLso haplotype A sequence (NZ1; NZ_JMTK01000001-5.1) used as the source of chromosomal gene sequences.d The CLso homologs identified from a Blast search of annotated CLas genes against the CLso the LsoNZ genome reference sequence (NC_014774.1)based on the tblastn algorithm.The predicted chromosomal coding region of the potato psyllid 'Central haplotype'associated Wolbachia endosymbiont, herein ' AZWo' , from the reference sequence, HGO53_RS03885 and WDIAC_RS0101550.f Predicted homolog of Wolbachia endosymbiont of Diaphorina citri (strain wDi; CP051608.1).g The genome reference Genbank Accession number is available for the Wolbachia endosymbiont of the potato psyllid, hence contigs from the laboratory-derived adult PoP transcriptome, which was the source of sequences used in this study (Brown Laboratory, The University of Arizona) was used for the ortholog searches.h A predicted Wolbachia repressor-like protein (WP_017531870) with hypothesized involvement in modulating the CLas prophage infection cycle in D. citri 46 .www.nature.com/scientificreports/ The PCR products were cloned into the pGEM®-T Easy Vector Kit (Promega, Madison, WI), according to the manufacturer's instructions.Ligation products were transformed into chemically competent Escherichia coli DH5α cells.Colonies were assayed by colony PCR amplification to identify those harboring the expected size insert.The plasmid vector containing a cloned fragment of the expected size was isolated from overnight cultures using the GeneJET Plasmid Miniprep Kit (Thermo Scientific).The concentration and purity of DNA plasmid were determined using the nanodrop spectrophotometer (ND-1000, NanoDrop Technologies, Wilmington, DE, USA).The inserts were sequenced bi-directionally (Sanger) (Eton Biosciences, Inc.) with universal primers, M13R and M13F.The sequences were assembled and annotated using the respective sequence database for CLso.

Isolation of RNA, cDNA synthesis, and reverse transcription
Based on the hypothesis that CLso-prophage gene expression is essential for survival and pathogenicity in both the plant and psyllid host, relative expression of CLso-prophage orthologs in the different PoP life stages and CLso-infected tomato leaves was quantified from total RNA purified from CLso-infected and CLso-free psyllids, or from CLso-infected or CLso-free (qRT-PCR negative control) tomato plants.
Total RNA was purified from groups of 25 teneral adults, 50 4th-5th (25 each) immature instars, and 1st-2nd (100 each) immature instars.Psyllids were transferred to a sterile microcentrifuge tube containing 1000 µl TRIzol® Reagent (Invitrogen, Carlsbad, CA) and one sterile 5 mm steel bead, and pulverized in a bead beater for 5 min.The total RNA was isolated according to the manufacturer's instructions.The RNA quantity and quality were determined based on the 230/260 and 260/280 ratios, respectively, using a Nano-Drop 2000 Spectrophotometer (Thermo Scientific, Wilmington, DE), with concentrations calculated by the A260/A280 ratio (~ 2.0).Quality and integrity were further evaluated by gel electrophoresis of a 2 μl sample on a 1.2% agarose gel in TAE buffer, pH 8.0, with visualization by GelRed (Biotium, USA) staining.
For tomato plants, the youngest fully expanded leaves (~ 100 mg) were collected from the same tomato plant (12-14 leaf-stage plants, 3-weeks post-inoculation) on which the respective psyllid cohort was reared.Leaves were washed with a stream of distilled water to remove psyllid instars and eggs to avoid possible contamination of RNA / cDNA synthesis.The tomato leaves were transferred to a centrifuge tube (2.0 ml), containing 1 ml TRIzol® Reagent.The total RNA was isolated as described above.
The RNA purified from psyllids or plants was diluted with nuclease-free water to a final concentration of 200 ng/µl, and treated with RNase-Free DNase (Invitrogen, Life Technologies, Carlsbad, USA), according to the manufacturer's instructions.Reverse transcription was carried out using 2 ug of total RNA per 20 μl reaction with the high-capacity cDNA reverse transcription kit (Applied Biosystems, Carlsbad, CA), according to the manufacturer's instructions.

Reference gene selection for qRT-PCR analysis
Previous analyses have documented the neutral expression of CLso recA 31,32 and 16S rDNA genes 33 , respectively, verifying their utility as reference genes for normalization of gene expression determined by qRT-PCR amplification in the potato psyllid-CLso pathosystem.The relative expression of CLso recA 31,32 and 16S rRNA were determined by qRT-PCR amplification, and Cq values were compared with CLso genome copy number (Cq), based on Pearson's correlation coefficient (PCC) 32 .The correlation coefficients for CLso genome copy number and recA and 16S rRNA gene expression were 0.94 and 0.80, respectively, indicating robust correlations (r > 0.95) between CLso genome copy in both immature and adult stages (Fig. 1), with recA expression being the most acceptable for all psyllid developmental stages.The recA gene was selected as the internal reference gene for normalization of gene expression.
analyzed for each biological replicate (n = 3), in parallel with a no-template (water) control, and a negative control containing no-reverse transcriptase.The primers and probe combinations for CLso and Wolbachia spp.mRNA quantification were designed using the IDT Assay Selection Tool available on the IDT website (S2 Table ).The PrimeTime® primers and probe were synthesized by Integrated DNA Technologies (IDT).The reaction efficiency for each primer/probe combination was determined by constructing a standard curve for a tenfold series of dilutions ranging from 1 × 10 9 to 1 × 10 2 copy number/μl using a cloned fragment of the respective target gene.The plasmid vectors harboring a cloned fragment of the expected size were purified using a Qiagen miniprep kit, linearized by endonuclease digestion (NEBcutter V2.0; http:// www.labto ols.us/ nebcu tter-v2-0/), and visualized on a 1% agarose gel.An undigested plasmid containing the respective cloned insert was included as a control to confirm endonuclease activity.The standard curve was derived by plotting the mean cycle quantification (Cq) of the tenfold serial dilutions of linearized plasmid DNA.The slope was used for estimating the reaction efficiency (E) based on the formula: E = 10^ (− 1/slope) − 1.
The RT-PCR amplification reactions (20 μl vol) were carried out in a 96-well plate using amplicon-specific TaqMan probe and primer combinations.Each reaction contained 10 μl of 2X TaqMan Universal Master Mix (Applied Biosystems), 1 μl 20 × Primetime primer/probe stock, 5 μl of RNase-free water, and 4 μl (100 ng/µl; 400 ng) of cDNA template).The qPCR cycling parameters consisted of 50 °C for 2 min initially, 95 °C for 10 min, and 40 cycles of a 2-step program (95 °C for 15 s, 60 °C for 60 s).Three negative control reactions containing no cDNA template were included for each run.
A combination of reference genes for normalization of expression, and a comparison of transcripts expressed in the dual CLso host-system consisting of tomato plants and psyllid vector-insect, the former in which all CLso genes and prophage loci are thought to be expressed, and the latter in which only some of the CLso-prophages are hypothesized to be expressed.
The relative expression of the predicted CLso chromosomal genes and prophage loci were quantified by qRT-PCR amplification from cDNA synthesized from mRNA isolated from immature and adult CLso-infected PoP and from plant CLso-infected tomato plants.The relative fold-change in the expression of the gene of interest was normalized to the recA (reference gene) and compared to the tomato sample (calibrator sample), using the formula 2 ΔΔCt34 where ΔΔCt = (C t-target − C t-reference) PoP sample − (C t-target − C t-reference) tomato-sample.Statistical significance (p < 0.05) between normalized expression of the gene of interest, relative to the expression of the analogous genes/loci in the tomato plant host was analyzed using the student's t-test implemented in CFX Maestro 1.1.v. 4.1.2433.1219software (Bio-Rad).The mean differences between the expression of the gene of interest at different PoP life stages were analyzed by ANOVA, and mean separation was based on Fisher's least significant difference (LSD) test (p < 0.05), with InfoStat® software, version 2020.

Accumulation of CLso in potato psyllid and tomato plants
The number of CLso genome copies was determined by qRT-PCR amplification of CLso 16S rRNA gene from total DNA isolated from cohorts consisting of 1,2 and 4, 5 nymphal instars, and teneral adult stages, and from tomato plants.Primer and probe design were according to a previously published method for PCR amplification of the CLso 16S rDNA gene 35,36 , present as three copies in the genome 15 .The primers and probe F-CLsoF-qPCR and R-CLso-16S-qPCR-P/HLBr-qPCR (Table S2), were used for real-time quantitative RT-PCR analysis of CLso 16S rRNA in total RNA purified from potato psyllids and tomato plants.The cloned linearized insert was diluted to a final concentration ranging from 10 1 to 10 10 in 2 or 40 ng/µl of CLso-free psyllids and uninfected tomato genomic DNA.A standard curve was established for each bacterial gene or phage locus to facilitate quantitative analysis.Copy number was calculated based on the average mass of a DNA base pair of 660 Daltons with the following formula, DNA (copies/ml) = DNA (ng/ml)/(DNA (bp) * 16,109 (ng/g) *660 (Da/bp)/6.02261023(copies/mole) 35 .The plasmid vector (~ 5 µl per plasmid with insert) and psyllid and tomato genomic DNA (10 ng psyllid or 200 ng tomato total genomic DNA, respectively) were used as templates for the respective qRT-PCR reaction.Each biological replicate was analyzed in triplicate and the CLso genome copy number was expressed, per 1000 ng of PoP or tomato genomic DNA, respectively.

Liberibacter genome copy in tomato plant and psyllid hosts
To explore the molecular mechanism of CLso propagation and circulation in a psyllid host, we measured the CLso bacterial accumulation (genome copy number) and the expression pattern of some related predicted genes associated with CLso propagation and circulation across the different PoP life stages.The CLso genome copy number in CLso-infected tomato plants and from psyllid 1st-2nd instars, 4th-5th instars, and teneral adults was determined by qRT-PCR amplification (Fig. S1).Results indicated an average of 3.0 × 10 5 , 2.7 × 10 3 , 6.4 × 10 5 and 2.1 × 10 6 copies/µl in total DNA in tomato plant, 1st-2nd, 4th-5th, teneral adult stages, respectively.Accumulation of CLso was significantly higher in the teneral adults and 4th-5th instars compared to the 1st-2nd instars (F = 17.8, df = 3, P-value = 0.0013).There was no significant difference in CLso accumulation (genome copy number) between 4th-5th instars and teneral adults (F = 6.12, df = 1, P-value = 0.06), which is consistent with previously published results 37 indicating CLso accumulated to lower levels in 1st and 2nd PoP instars, compared to 4th-5th nymphal or adult stage, respectively.

Expression of CLso orthologs and prophage loci
To investigate if CLso genes are differentially expressed during the different psyllid development stages, the expression of selected CLso-prophage genes/loci was quantified for the different PoP stages, and results were compared to the expression of the analogous genes/loci in the tomato plant host.Thirty-four predicted CLso orthologs that consisted of 13 CLso-A haplotype chromosomal genes, 20 predicted prophage loci, and one www.nature.com/scientificreports/predicted Wolbachia spp.chromosomal genes were analyzed for relative gene expression for pooled samples of PoP young 1st-2nd, 4th -5th, and teneral adults (Table 1).Five of 34 CLso predicted orthologs analyzed for relative expression exhibited increased expression in the psyllid host, compared to the tomato plant, while 13 of the 34 genes exhibited reduced expression, independent of psyllid life stage, and 15 CLso coding regions were differentially expressed to some extent for all PoP stages examined here (Table 1).
The relative expression of CLso selected genes ranged from ~ 5.97 log2FC (equal to 63-fold-change) for CLso-ortholog of flagellin, FliC (AZch6) to 6.6 times lower (equal to -90 times fold-change) for CLso-ortholog of glutathione peroxidase (AZph9) compared to expression of the analogous CLso genes in the tomato plant host.

Orthologs associated with CLso motility, adhesion, and/or attachment
To determine the potential involvement of CLso-predicted pili and flagellar genes in infection of the insect vector and tomato plant host, the expression of pili-and flagellar-genes were analyzed for the different PoP life stages and compared with the expression of the analogous CLso genes in the tomato plant host.The orthologs predicted to encode pili (AZch8) and flagellar (AZch6; AZch7) proteins exhibited increased expression in PoP compared to CLso-expression of the analogous loci detected in CLso-infected tomato plants.Expression of predicted flagellarassociated proteins, FliC (F = 377.5,df = 2, P-value < 0.001), and (F = 887.8,df = 2, P-value < 0.001), was high in teneral adults, compared to moderate and low in 4th-5th and 1st-2nd instars, respectively.In contrast, the CLso predicted Flp3 encoding putative pilus protein (AZch8) was more highly expressed in the first and second instars than in the 4th and 5th instars or teneral adults (F = 161.9,df = 2, P-value < 0.001) (Fig. 2, Table 2).

CLso ortholog of prophage genes associated with potential lytic cycle, phage cycle regulation, and lysogenic conversion in immature and adult potato psyllids
To determine if the expression of CLso-prophage loci with predicted involvement in lytic and lysogenic infection cycles were indicative of a prophage lytic cycle-induction hypothesis, the expression of selected prophage loci was quantified by qRT-PCR in the psyllid and tomato plant host, respectively.Of particular interest was to determine if the SC1 and SC2 prophages were stably integrated or conversely, activated lysogens, respectively.The CLso prophage orthologs of SC1-and SC2-prophages have been previously predicted to encode lysis, structural, phage cycle regulation (transcription factor), and lysogenic conversion functions (Table 2, Fig. 3).Analysis of prophage genes (orthologs) associated with the lytic cycle, and the predicted structural-(AZph1, AZph7), lysis-(AZph2), and DNA replication-(AZph17) functions, indicated that their gene expression was reduced in all PoP stages, compared to expression in planta (Table 2, Fig. 3).Gene expression was significantly different between the teneral adults and nymphal stages for AZph2 (F = 12.4,df = 2, P-value = 0.0074), the loci encoding predicted endolysin and AZph7, predicted major capsid protein (F = 330.5,df = 2, P-value < 0.001), whereas, no differences (F = 1.38, df = 2, P-value = 0.3208) in expression of AZph1 (predicted phage structural protein) were observed for any PoP stages.Finally, significantly decreased expression of the predicted DNA polymerase A, AZph17, was observed in all PoP developmental stages (Table 2, Fig. 3) compared to the expression level observed in tomato plants (F = 208.1,df = 3, P-value < 0.001).
The CLso ortholog locus encoding the predicted prophage anti-repressors, AZph14 (F = 224.6,df = 3, P-value < 0.001), AZph15 (F = 34.5, df = 2, P-value < 0.001), and AZph16 (F = 55.5, df = 3, P-value < 0.001), was expressed at low levels in the nymphal and teneral adults, compared to in planta expression.However, the AZph11 locus, encoding the predicted phage-related repressor protein C2, was highly expressed in all psyllid stages, Table 2. Relative expression of selected "Ca.Liberibacter solanacearum" (CLso) and Wolbachia gene (s) in CLso-infected psyllid stages compared to expression of the analogous gene in CLso-infected tomato leaves and CLso-free psyllid stages, respectively. 1Loci analyzed for "Candidatus Liberibacter solanacearum" (CLso) haplotype A and were designated "AZph #" and "AZch#" for the prophage and chromosomal genes, respectively.Loci analyzed for the Wolbachia spp.endosymbiont of Bactericera cockerelli potato psyllid (PoP) 'Central haplotype' were designated "AZWo". 2 Fold-change (log2 ratio) relative gene expression in CLsoinfected psyllid stages, compared to CLso-infected tomato plant and CLso-free psyllid stages, included as references for CLso and the Wolbachia endosymbiont of B. cockerelli, respectively.Data were normalized to values for the CLso chromosomal reference gene recA, and Wolbachia spp.FtsZ, respectively.The mean values indicated by the same letter are not significantly different (p > 0.05).Values representing positive fold-change (log2 ratio) are indicated in bold.ϕ = No significant difference between normalized expression of the gene of interest, relative to the expression of the analogous gene/locus in the control sample was determined based on the student's t-test at p > 0.05.compared to the CLso-infected tomato plant host.Gene expression was higher in the 4th-5th instars and teneral adults, compared to the youngest instars, 1 and 2 (F = 57.19,df = 2, P-value < 0.001) (Table 2).No significant difference (p > 0.05), in expression CLso locus AZph10 encoding the predicted phage-related repressor protein C20 (F = 0.48, df = 3, P-value = 0.6429) was observed for any of the psyllid stages analyzed here, compared to gene expression in the tomato plant host.Interestingly, the analogous CLso chromosomal locus encoding the predicted bacteriophage repressor protein C1 (AZch1) was highly expressed (F = 4.3, df = 3, P-value = 0.0459) in all potato psyllid life stages (Fig. 3).The relative expression of the AZph5 phage locus, a predicted integrase/recombinase involved in phage DNA integration, was consistently low for all life stages of PoP (F = 34.27,df = 3, P-value < 0.001) (Table 2).However, nymphal instars 1 and 2 showed greater expression of the integrase/ recombinase (AZph6) predicted locus, compared to the tomato plant host (F = 18.37, df = 1, P-value = 0.0011) while 4th-5th instar nymphs and teneral adults showed lower expression level compared to its gene expression in the tomato plant host (F = 13.25, df = 2, P-value = 0.0063) (Table 2) Expression of CLso orthologs encoding RecB-like nuclease, AZph12, and AZph13, which are predicted recombination repair enzymes, and expression of AZph6 were comparable, suggesting they are potential interactors, and so are likely temporarily expressed (Table 2, Fig. 3).The CLso-A haplotype homologs of CLas-prophage loci AZph8 and AZph9, encoding the predicted lysogenic conversion factor, peroxidase, were expressed in all PoP stages at a significantly lower level than the analogous loci expressed in the CLso-infected tomato plants (AZph8: F = 182.53,df = 3, P-value < 0.001; AZph9: F = 229.14,df = 3, P-value < 0.001) (Table 2).Similarly, the AZph3 and AZph4 loci encoding a predicted toxin-antitoxin system (TA) with predicted colicin IA-like functions, and the predicted cognate colicin immunity protein encoded Figure 3. Relative expression of "Candidatus Liberibacter solanacearum" (CLso) prophage loci in different potato psyllid life stages.Relative gene expression was calculated using the formula ΔΔCt for real-time quantitative analysis of transcripts detected in the different CLso-infected potato psyllid life stages (insect host), relative to the analogous prophage loci expressed in CLso-infected tomato plants (plant host).Results were normalized using the CLso-chromosomal reference gene recA.The error bar represents the standard error of the mean.Different letters indicate a statistically significant difference in expression between the psyllid life stages for which the same gene was analyzed (ANOVA with Fisher's LSD test, P-value < 0.05).

CLso and prophage gene expression associated with transport of effectors with predicted involvement in virulence and pathogenicity
To investigate selected CLso-prophage genes with predicted functions in "Ca.Liberibacter" virulence and pathogenicity, gene expression was quantified for CLso orthologs encoding the predicted metalloprotease serralysin domain, TolC, Imelysin, LuxR, Liberibacter' genus-specific secretion protein, and serine/tyrosine phosphatase, in the different PoP life stages and results were compared to expression of the analogous orthologs in the tomato host plant.The higher expression of CLso ortholog encoding the predicted secreted metalloprotease serralysin domain (AZch4) was observed in the nymphal stages compared to the teneral adults (F = 21.06,df = 2, P-value = < 0.001).In contrast, relative expression of CLso TolC protein (AZch9) ortholog, the third predicted component of the T1SS I secretion system, was elevated in the 1st-2nd compared to the other PoP stages examined (F = 176.57,df = 2, P-value < 0.001) (Table 2, Fig. 3).Expression of a CLso Imelysin domain protein ortholog (AZch5), a predicted secreted protein 14 was higher in all PoP stages, compared to the tomato plant host, and relative expression increased significantly, according to the respective PoP instar developmental stage(s) (F = 1050.05,df = 3, P-value = < 0.001) (Table 2, Fig. 2).Similarly, expression of the CLso ortholog of the locus encoding LuxR (AZch2) was differentially expressed in the mature (4th-5th) instars and teneral adults of PoP, compared to younger 1st-2nd nymph stages (F = 137.44,df = 2, P-value < 0.001) (Table 2, Fig. 2).Expression of the predicted CLso LuxR gene was found to increase dramatically in both 4th-5th instars (~ 5 times, fold change) and teneral adults (~ 13-fold change), but not in the 1st-2nd stage of PoP (Fig. 2, Table 2).Expression of the CLso homolog of the predicted 'Liberibacter' genus-specific secretion protein (LUSP) 16 , AZch10, increased in teneral adults (F = 29.17,df = 1, P-value = 0.005), compared to the PoP 4th-5th instars, for which no significant differential expression was observed (F = 0.36, df = 1, P-value = 0.58) relative to expression of the analogous genes in the tomato host plant (Table 2, Fig. 2).

Discussion
To establish and maintain compatible associations with divergent hosts that require a dual host strategy, Liberibacter spp.must be capable of responding to host-specific signals.Alternative lifestyles and colonization strategies in different host species may therefore be associated with a consequence of such adaptation to two different host environments.In this study, the expression pattern of 33 CLso-prophage genes/loci for the different PoP life stages was characterized and compared with the expression of analogous genes/loci (when expressed) in the CLso-infected tomato plants to investigate if Liberibacter has adopted different strategies to facilitate colonization of the insect (animal) compared to the plant host, and also if interactions differ among the different psyllid life stages when associated with each host, respectively.
Quantification of CLso copy number revealed that bacterial accumulation was significantly higher in the teneral adults and 4th-5th instars, compared to the 1st-2nd instars.No significant difference was observed in CLso accumulation (copy number) between 4th-5th instars and teneral adults.These observations are consistent with previously published results 37 indicating that CLso genome copy number was lower in the 1st and 2nd instars, compared to the 4th-5th nymphal or adult stages, respectively.Similar levels of gene expression were for the older instars (4th-5th) and teneral adults, with the majority being prophage genes, underscoring important differences especially in prophage activity among the different developmental stages of psyllids (which consist of five nymphal instars, and the adult stage) (Table 1).Previous studies have shown that certain Liberibacter genes are expressed in the psyllid and/or in the plant host, or are predicted to be host-specific 13,14,22,38,39 .Also, differential expression has been reported among the different psyllid stages, and by the plant and psyllid host, respectively 8,9,32 , leading to the hypothesis that different genes may be essential for CLso adaptation and pathogenicity within these different host 'environments' i.e. animal versus plant 40 .
The expression of predicted CLso gene encoding pili and flagellar assembly proteins revealed that pilin expression in younger psyllid instars was higher than in all other psyllid life stages, while flagellin, and FlgL were more highly expressed in the older immature instars and adult stages, compared to the younger instars.This appears to reflect differences in biological function and/or differences in temporal expression in CLso host developmental stages, which would be consistent with the culmination of the infection cycle in teneral adults to facilitate optimal transmission to the host plant by those teneral adults dispersing to colonize a new host where reproduction will occur.Such patterns of gene expression are consistent with previously recognized functions of bacterial pili and flagella in adhesion and motility, respectively 4,41 .Increased expression of genes associated with the flagellar assembly (flgL, flagellin) and pilin proteins (Flp/Fap) has been reported in the psyllid gut and whole body expression libraries 8,14,32,38 , indicating host attachment utilizing "Ca Liberibacter"-encoded surface appendages is integral to psyllid host infection and probably to motility, post-exocytosis from gut cells into the hemolymph and during circulation to salivary glands.Both flagella and a Type IV pilus (T4P) are known to contribute to bacterial pathogenicity in general, and also in fastidious "Ca.Liberibacter" species 15,16 .Previous studies of the CLso-PoP pathosystem have hypothesized a direct involvement of the T4P system in virulence and pathogenesis 24 , specifically, through secretion of "Ca.Liberibacter" effector(s) into the host cell.Also, the T4P system is involved in host cell attachment pathosystems 42 and biofilm formation 43 .In the potato psyllid, dense biofilms have been observed on the gut surface, and filter chamber of 3-5th instars, and adults 4 with the greatest densities associated with the 4th and 5th instars, and teneral (young) adults 4 .Further, flagella-like surface appendages have been observed in PoP alimentary canals using transmission electron microscopy (TEM) 44 , however, flagellated "Ca.Liberibacter" cells have not been reported in planta 2 .
Expression of CLso ortholog-prophage loci with a predicted role in lytic cycle induction was lower in PoP stages, compared to that in CLso-infected tomato plants.However, other genes associated with the lysogenic cycle showed increased expression in PoP adults, and in all of the immature PoP psyllid instars analyzed here.These results suggest that lysogeny occurs in the PoP host and that most probably the prophage proteins are also involved in lysogenic conversion and induction of a lytic cycle in the tomato plant host.In this study, and in previously published reports 14,18,32,45,46 , the "Ca.Liberibacter" (ACP-CLas) lytic cycle is activated primarily, if not only, in planta.In PoP, lysogenic-lytic cycling of CLas was negligible or undetectable, thereby suggesting the involvement of a predicted repressor-anti-repressor regulatory system, well-known for other bacterial pathosystems to activate the lytic cycle.Although the lysogenic cycle appears to operate in both the tomato plant and PoP host, the lysogenic stage appears to occur overwhelmingly in the tomato plant host, suggesting that 'leaky" repression may occur toward the end of the infection cycle in the psyllid host 47 .Expression of a small Wolbachia-encoded protein was previously hypothesized to regulate the CLas phage lytic cycle in the ACP host 46 by repressing expression of the holin gene in the lytic operon 16 .However, here there was no evidence that a putatively homologous Wolbachia-encoded protein was involved in the PoP-CLso infection cycle, as was previously hypothesized for the ACP-CLas pathosystem 46 in which host-specific expression profiles were implicated in host-switching.To test this hypothesis, the relative gene expression of a predicted homolog of the putative Wolbachia repressor was evaluated for different CLso-infected PoP stage compared to the respective CLso-free PoP samples.The PoP-Wolbachia spp.repressor protein ortholog 46 was either undetectable or expressed at undetectable low levels in CLso-infected PoP immature and teneral adult stages, respectively, compared to expression in CLso-free PoP (data not showed).This result suggests that the putative Wolbachia-encoded repressor is not differentially expressed at a detectable level and/or that the repressor is not involved directly in CLso infection of PoP.Also, the nucleotide and protein blast searches failed to identify CLas holin orthologs among CLso genomes available in GenBank, suggesting that ACP and PoP-associated Wolbachia spp.may contribute differently to the psyllid (animal) and plant host interactions in this dual-host pathosystem.Notably, the CLso-predicted prophage repressor proteins, AZch1 and SC2_ AZph11, were expressed in adult and immature PoP stages, suggesting that the repressor protein is integral to maintaining the lysogenic cycle in the psyllid host, suggesting there is no need for a Wolbachia-like repressor protein in PoP 47 , even though an analogous protein has been implicated in the ACP-CLas pathosystem.
Another predicted repressor, the bacteriophage cI repressor, exhibits variation in tandem repeat number (VNTRs) and frequency among CLas-predicted SC1-and SC2-prophage and CLas-VNTR bacteriophage cI repressor proteins 48 .Further, CLso-prophage-encoded predicted integrase mediates lysogeny through the integration of phage DNA into the bacterial genome 49 .Here, the observed increased expression of CLso orthologs of prophage loci involved in DNA integration (AZph6) and recombination repair (AZph12; AZph13) has provided evidence for prophage DNA integrated in the CLso genome during infection of early immature PoP instar(s), which has been linked to the PoP stages in which the immune system is least compromised 8 .Further, a Cas4 protein is located within the SC1/2_gp195 loci (CLso orthologs of AZph12 and AZph13) (Zheng et al. 2016).This suggests that the Liberibacter CRISPR/Cas system may possibly contribute to superinfection and/or immunity through lysogen expression in the psyllid host.
The higher expression of CLso ortholog of lysogenic conversion genes that encode the peroxidases and TA system implicates their involvement as lysogenic conversion factors that are associated with ''Ca.Liberibacter'' pathogenicity in plant host 22,50 , and is consistent with the results of a previous study in which they were predicted to serve as bacterial virulence effectors that suppress host plant immunity 18 .These phage loci may represent examples of genes expressed in both hosts of the ''Ca.Liberibacter''-psyllid pathosystem, albeit at different levels and/or possibly, temporally, in the context of the insect and plant infection cycle, respectively.
The results also provided evidence of host-specific expression patterns for several candidate effectors, including Imelysin, for whose expression was also observed in the PoP-CLso pathosystem for all life stages, as well as for the ABC transporter/ATPase, LysE family translocator, and DsbA-like protein, for which expression was higher in tomato plants, compared to the psyllid host, respectively.Also, PoP stage-specific expression was documented for TolC, serralysin, LUSP, and LuxR, genes with predicted involvement in virulence and pathogenicity.In PoP, TolC and serralysin expression levels were similar to those reported for ACP, as well as their higher expression in early immature stages.These observations are consistent with the in silico predictions that serralysin is a TISS substrate 15 .Previous studies have reported that serralysin is expressed in CLas-infected citrus 14 and in CLso-infected PoP 51 , respectively.Serralysin-like proteins are known to be produced by other plant and human pathogenic bacteria 14 to aid in circumventing host antimicrobial defenses by inactivating antimicrobial proteins and peptides 52 .Consistent with reduced serralysin expression of predicted immune system-related genes in PoP nymphs (62%) and adults (43%) 8,9 , shifts in defense response genes expression in adult PoP may be attributable to increased expression of developmentally-associated serralysin, a well-characterized virulence factor.Thus, CLso appears to capitalize on the "weak" immune response observed for first through third nymphal instars early in the infection cycle 8,9 .
Vol:.( 1234567890 www.nature.com/scientificreports/Consistent with CLso Imelysin transcript levels, CLso accumulation increased concomitantly during maturation of the psyllid host i.e. in the early adulthood stage (Fig. 3, Fig. S1).These results support the possible involvement of a predicted Imelysin-like protein in the uptake of iron from the potato psyllid host by the CLso pathogen, which is consistent with knowledge that Imelysins-belong to a superfamily of bacterial proteins involved in iron uptake 53 .
Up-regulation of LUSP in the PoP teneral stage reveals their probable roles in Liberibacter-psyllid interaction in a time-and/or stage-specific manner.CLso has one copy of these unknown proteins while four homologs of LUSP are predicted in the CLas genome 16 .The LUSP-like proteins may harbor signal peptides (SPs), which prompt cells to translocate proteins to the cell membrane, however, no homolog has been identified by in silico analysis of available microbial databases, suggesting this protein functions as a "Ca.Liberibacter"-specific virulence factor 16 .
The plant host-specific expression patterns of CLso orthologs for serine/tyrosine phosphatase, ABC transporter /ATPase, LysE family translocator, and DsbA-like protein were consistent with their involvement in adaptation and survival within the plant but not necessarily in the psyllid (animal) host, respectively.The virulence functions predictions include interfering with the host immune response through protein kinase signaling cascades 16 , uptake and export nutrients e.g., amino acids, lipids, and heavy metal ions 54,55 and protein secretion 56 .
Members of LuxR family proteins regulate gene expression during quorum sensing to regulate bacterial behaviors, including symbiosis, motility, biofilm formation, and virulence 57,58 , and are involved in insect vector transmission of the bacterium Xylella fastidiosa 59 .The coordinated expression of CLso ortholog of predicted LuxR and other studied genes associated with motility and virulence observed herein (Fig. 2) suggest that the LuxR transcriptional factor family, may coordinate a density-dependent switch in expression of Liberibacter effectors that provide motility, biofilm formation, and virulence functions.
Here, the developmental stage-specific expression of the CLso-prophage homologs (to CLas), auto-transporters SC2_gp240 (AZph19) 18 and LasAI (AZph20) 60 , the latter, involved in virulence/transport, was higher in the youngest immature instars (Table 2, Fig. 3).In addition to SDEs, auto-transporters rely on the Sec machinery for transport in "Ca.Liberibacter" 18,61 .These loci encode predicted proteins harboring tandem repeats that share homology with proteins identified as leucine-rich repeat-, cell wall-associated biofilm-, and cell surfaceproteins 18,61 .The LasAI gene, an auto-transporter 60 , has been implicated in "Ca.Liberibacter" acquisition in the psyllid salivary glands 18 .The role of auto-transporters in pathogenic "Ca.Liberibacter" species is unknown; however, it seems that they may use an auto-transporter type V secretion system (T5SS) as a substitute secretion system for either a canonical T3SS or T4SS 60 .The increased expression of the predicted auto-transporters, AZph19 and AZph20, in PoP immature instars, suggests that the latter proteins are indispensable for adhesion and colonization that is known to occur early in the CLso-PoP infection cycle, based on this and previous studies of this pathosystem.

Conclusion
The fastidious nature of "Ca.Liberibacter" spp.has limited the understanding of the interactions of Liberibacter spp.The availability of bacterial and psyllid vector genome and transcriptome sequence databases for analyses has aided in the discovery of co-evolved "Ca.Liberibacter"-psyllid and -plant host effectors crucial for establishing interkingdom interactions and short and/or long-term survival modes in divergent hosts 8,9,14,31,32 .The results reported here reinforce previous functional genomics-derived hypotheses that the cross-kingdom adaptation of "Ca.Liberibacter" will require at least some host-specific bacterial-and prophage-encoded genes, expressed in one host or the other, and/or whose expression is temporally up-or down-regulated in both hosts and in the context of host developmental stage.Prominent among Liberibacter chromosomally-encoded genes were effectors with predicted functions in pathogenicity, including cell wall adhesion-, transporter-, iron-scavenging-, and toxin-anti-toxin-functions, and thiol-disulfide oxidoreductase-like activity (disulfide bond formation motif), previously associated with other bacterial-secreted toxins, virulence factors, adhesion machinery, and motility structures.Also notable were peptidase, phosphatase, and protease activities during early-, mid-, or late processes of CLso-psyllid host invasion, as well as expression of the CLso-prophage autotransporter adhesion protein in the psyllid host, feasibly functioning in gut cell wall adhesion.
This study presents the first in depth analysis of the PoP-CLso-prophage pathosystem that is associated with an herbaceous plant host of CLso.The results underscore similarities and differences with the better-studied ACP-CLas pathosystem, associated with a woody perennial plant host.In both study systems (PoP-CLso, ACP-CLas), "Ca.Liberibacter" gene and phage loci expression appear to modulate gene expression of the respective psyllid vector/host, potentially, to some extent through co-coordination of expression of the co-interacting elements of the pathosystem.These results have revealed remarkably fine-tuned, developmental stage-specific gene expression, seemingly orchestrated to facilitate the multiplication of the CLso pathogen to extraordinary levels in the psyllid host, without resultant mortality, or at least, until the bacterial pathogen has been transmitted to its' plant host.

Figure 2 .
Figure 2. Relative expression of "Candidatus Liberibacter solanacearum" (CLso) selected genes in different Bactericera cockerelli potato psyllid (PoP) stages, compared to the analogous genes. in CLso-infected tomato plants.. Data were normalized by comparison with the CLso-chromosomal reference gene recA.Error bars represent the standard error of the mean.The different letters indicate a statistically significant difference in expression between the psyllid life stages for which the same gene was analyzed (ANOVA with Fisher's LSD test, P-value < 0.05).